Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to des...Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to design primer probes covering 24 p72 genotypes. The optimal amount of dimethylsulphoxide (DMSO) for qPCR amplification was determined, Various sensitivity and limit of detection (LOD) tests were performed, and clinical samples from China and imported goods were tested. Results The optimal primer-probe combination could specifically detect ASFV, 1.5% DMSO was optimal for qPCR, and LOD reached 3.2 copies/μL with good reproducibility (n = 20, p = 0.369). The method was employed to test 142 clinically suspected samples, of which 30 pig blood and 37 pig tissue samples were ASFV-positive. Moreover, the positive testing rate for ASFV was higher than for the standard qPCR method recommended by the Office International Des Epizooties (OIE), and for the commercially available kit. Thus, our method is superior for testing weakly positive samples with low virus titre, and epidemic strains present in imported goods. Conclusion Our method could be employed for universal testing of epidemic ASFV strains worldwide, ensuring wider coverage of hosts and ASFV strains/endemic strains, reducing false<span style="font-family:;" "=""> </span><span style="font-family:Verdana;">negatives, and benefitting early diagnosis.</span>展开更多
金黄色葡萄球菌是花生及其制品中常见的一种食源性致病菌,针对金黄色葡萄球菌的快速检测方法对实现花生及其制品中的食品安全防控有重要意义.根据金黄色葡萄球菌的耐热核酸酶基因(nuc)设计了3对特异性引物,同时优化了退火温度,成功建立...金黄色葡萄球菌是花生及其制品中常见的一种食源性致病菌,针对金黄色葡萄球菌的快速检测方法对实现花生及其制品中的食品安全防控有重要意义.根据金黄色葡萄球菌的耐热核酸酶基因(nuc)设计了3对特异性引物,同时优化了退火温度,成功建立了一种特异性引物的SYBR Green实时荧光定量PCR(qPCR)快速检测方法,进一步对该方法的特异性、重现性及灵敏度进行了验证.以8种模拟污染花生加工产品为实验对象,对该方法与荧光定量PCR试剂盒-探针法及商品化Baird-Parker干粉培养基法进行了评价研究.结果表明,建立的SYBR Green qPCR快速检测方法特异性和重现性良好,最低检测限为8.99 copies/μL,其灵敏度比荧光定量PCR试剂盒-探针法高2个数量级.SYBR Green qPCR快速检测方法具有成本低、检测快速、特异性好、灵敏度高的优点,可用于花生及其制品中金黄色葡萄球菌的快速检测,也可为防控其他食品中的金黄色葡萄球菌污染提供参考.展开更多
Helicobacter pylori (H. pylori) is bacteria considered to be present in half of the population and it is a public health problem worldwide. Most patients infected with H. pylori show no clinical symptoms;nonetheless, ...Helicobacter pylori (H. pylori) is bacteria considered to be present in half of the population and it is a public health problem worldwide. Most patients infected with H. pylori show no clinical symptoms;nonetheless, approximately 10% to 20% of these patients will develop peptic ulcers and 1% will develop gastric cancer. The International Agency for Research on Cancer has classified H. pylori as a Group 1 carcinogen, recognized as the only bacteria capable of producing cancer. Samples of drinking water (n = 44) from aqueducts with chlorination treatment in selected areas with high prevalence of gastric cancer were analyzed in Costa Rica. Samples of drinking water from Panamá (n = 44) from aqueducts supplying untreated water for human consumption in the province of Chiriquí were also analyzed. The molecular marker of H. pylori, glmM, was used, and to optimize the Real Time PCR (qPCR) technique, annealing temperature, concentration of primers and probe were standardized;also, by analyzing different standard curves, the best reaction conditions that allowed detecting and quantifying the gene were determined. The LightCycler® 480 II (LC480II) equipment from Roche Diagnostics GmbH was used, as well as the Absolute Quantification Analysis by means of the Second Derivative Maximum Method. In the case of the samples from Costa Rica, it was determined that 79.5% were positive for H. pylori;removing outlier high average, quantification of bacteria was determined in 3.6 × 103 copies/100 mL. For Panamá it was determined that 86% of the samples were found positive for the presence of H. pylori;removing outlier high average quantification of bacteria was determined at 3.3 × 102 copies/100 mL. The difference in values between the aqueducts in both countries revealed an environmental distribution of the bacteria of epidemiological interest in each case.展开更多
文摘目的本研究旨在建立一种实时荧光定量PCR方法,用于检测猕猴三磷酸腺苷结合盒转运蛋白G2(adenosine triphosphate-binding cassette transporter protein G2,ABCG2)mRNA的基因转录水平。方法使用NCBI上GenBank数据库猕猴(Macaca mulatta)的ABCG2核苷酸序列号NM_001032919.1及内参GAPDH核苷酸序列号NM_001195426.1,借助Primer premier 5.0软件设计PCR引物。提取猕猴新鲜肾组织的总RNA,并反转录合成cDNA。接着,利用PCR引物进行实时荧光定量PCR扩增,并根据反应体系中荧光的变化情况定量分析ABCG2的mRNA相对表达水平。结果PCR产物测序结果显示,扩增的ABCG2和GAPDH核苷酸序列与NCBI上猕猴的序列同源性分别为90.91%和91.14%。ABCG2和GAPDH的扩增效率均达到80%~120%,实时荧光定量PCR标准曲线的熔解曲线为单峰,R2接近1。结论本研究建立的检测猕猴ABCG2 mRNA实时荧光定量检测方法,为研究高尿酸血症的发病机制以及新药开发奠定基础。
文摘Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to design primer probes covering 24 p72 genotypes. The optimal amount of dimethylsulphoxide (DMSO) for qPCR amplification was determined, Various sensitivity and limit of detection (LOD) tests were performed, and clinical samples from China and imported goods were tested. Results The optimal primer-probe combination could specifically detect ASFV, 1.5% DMSO was optimal for qPCR, and LOD reached 3.2 copies/μL with good reproducibility (n = 20, p = 0.369). The method was employed to test 142 clinically suspected samples, of which 30 pig blood and 37 pig tissue samples were ASFV-positive. Moreover, the positive testing rate for ASFV was higher than for the standard qPCR method recommended by the Office International Des Epizooties (OIE), and for the commercially available kit. Thus, our method is superior for testing weakly positive samples with low virus titre, and epidemic strains present in imported goods. Conclusion Our method could be employed for universal testing of epidemic ASFV strains worldwide, ensuring wider coverage of hosts and ASFV strains/endemic strains, reducing false<span style="font-family:;" "=""> </span><span style="font-family:Verdana;">negatives, and benefitting early diagnosis.</span>
文摘金黄色葡萄球菌是花生及其制品中常见的一种食源性致病菌,针对金黄色葡萄球菌的快速检测方法对实现花生及其制品中的食品安全防控有重要意义.根据金黄色葡萄球菌的耐热核酸酶基因(nuc)设计了3对特异性引物,同时优化了退火温度,成功建立了一种特异性引物的SYBR Green实时荧光定量PCR(qPCR)快速检测方法,进一步对该方法的特异性、重现性及灵敏度进行了验证.以8种模拟污染花生加工产品为实验对象,对该方法与荧光定量PCR试剂盒-探针法及商品化Baird-Parker干粉培养基法进行了评价研究.结果表明,建立的SYBR Green qPCR快速检测方法特异性和重现性良好,最低检测限为8.99 copies/μL,其灵敏度比荧光定量PCR试剂盒-探针法高2个数量级.SYBR Green qPCR快速检测方法具有成本低、检测快速、特异性好、灵敏度高的优点,可用于花生及其制品中金黄色葡萄球菌的快速检测,也可为防控其他食品中的金黄色葡萄球菌污染提供参考.
文摘Helicobacter pylori (H. pylori) is bacteria considered to be present in half of the population and it is a public health problem worldwide. Most patients infected with H. pylori show no clinical symptoms;nonetheless, approximately 10% to 20% of these patients will develop peptic ulcers and 1% will develop gastric cancer. The International Agency for Research on Cancer has classified H. pylori as a Group 1 carcinogen, recognized as the only bacteria capable of producing cancer. Samples of drinking water (n = 44) from aqueducts with chlorination treatment in selected areas with high prevalence of gastric cancer were analyzed in Costa Rica. Samples of drinking water from Panamá (n = 44) from aqueducts supplying untreated water for human consumption in the province of Chiriquí were also analyzed. The molecular marker of H. pylori, glmM, was used, and to optimize the Real Time PCR (qPCR) technique, annealing temperature, concentration of primers and probe were standardized;also, by analyzing different standard curves, the best reaction conditions that allowed detecting and quantifying the gene were determined. The LightCycler® 480 II (LC480II) equipment from Roche Diagnostics GmbH was used, as well as the Absolute Quantification Analysis by means of the Second Derivative Maximum Method. In the case of the samples from Costa Rica, it was determined that 79.5% were positive for H. pylori;removing outlier high average, quantification of bacteria was determined in 3.6 × 103 copies/100 mL. For Panamá it was determined that 86% of the samples were found positive for the presence of H. pylori;removing outlier high average quantification of bacteria was determined at 3.3 × 102 copies/100 mL. The difference in values between the aqueducts in both countries revealed an environmental distribution of the bacteria of epidemiological interest in each case.
